ABSTRACT:

Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults, with median patient survival of just 14.6 months from the time of diagnosis. The current standard of care is surgery followed by radiation with concomitant chemotherapy; however, recurrence is inevitable and nearly 100% of patients ultimately succumb to their disease. A growing body of evidence has attributed GBM initiation and recurrence to a rare subpopulation of tumor cells characterized by an aggressive stem cell-like phenotype, called glioblastoma stem cells (GSCs). GSCs are highly refractory to radiation and chemotherapy, and they have been demonstrated to repopulate the tumor following treatment. Therefore, new strategies are desperately needed to eradicate both GSCs and differentiated, bulk GBM cells to improve patient outcomes. One potential way this may be achieved is by using RNA interference (RNAi) to suppress the expression of genes that drive GBM progression. Towards this goal, this thesis examines the role that Gli1, a key mediator of Hedgehog signaling, plays in the progression and chemotherapy resistance of GBM, and it develops a new type of polycation-nanoparticle hybrid to suppress Gli1 expression in GBM cells as a strategy for GBM therapy. This is accomplished through three objectives.

First, this thesis investigates the utility of suppressing Hedgehog (Hh)/Gli1 signaling as an adjuvant to temozolomide (TMZ) chemotherapy using in vitro models of GBM. We found that small interfering RNA (siRNA) against Gli1 could reduce GBM cell proliferation, metabolic activity, and drug efflux activity. Surprisingly, silencing Gli1 without TMZ co-treatment did not induce apoptosis in GBM cells; rather, it induced senescence in a loss of PTEN-depended manner. Excitingly, this work showed that pharmacological Gli inhibition in combination with TMZ could induce apoptosis in stem-like GBM cells grown as neurospheres. These results demonstrate that targeting Hh/Gli1 signaling offers a promising strategy to reduce the chemoresistance of GBM cells, and therefore we concluded that Gli1 is a potent target for therapeutic manipulation by siRNA nanocarriers.

While siRNA-mediated gene regulation has emerged a promising strategy to halt tumor growth, clinical translation of RNAi remains challenging because siRNA is rapidly cleared from circulation, easily degraded by nucleases that are present in physiological conditions, and cannot passively cross cell membranes due to its large size and negative charge. Nanoparticle-based siRNA carriers have been made to overcome these issues, but their translation is hindered by limited knowledge regarding the parameters that regulate their interactions with biological systems. To address this, we investigated the influence of polycation-based nanocarrier architecture on intracellular siRNA delivery. The cellular interactions of two polycation-based siRNA carriers with different siRNA orientation were compared: (1) polyethylenimine-coated spherical nucleic acids (PEI-SNAs), in which polyethylenimine is wrapped around a spherical nucleic acid core containing radially-oriented siRNA, and (2) randomly assembled polyethylenimine-siRNA polyplexes that lack controlled architecture. PEI-SNAs outperformed PEI-siRNA polyplexes in terms of net cellular uptake, lysosomal evasion, gene regulation potency, and cytocompatibility. These results highlight the importance of siRNA architecture in efficient nanocarrier design, and prompted us to explore the utility of PEI-SNAs targeting Gli1 as a therapy for GBM.

In the final objective of this thesis, PEI-SNAs targeting Gli1 were developed and evaluated against GBM cells. We found that Gli1 PEI-SNAs successfully silence tumor-promoting Hedgehog pathway genes and downstream target genes that promote the chemoresistant phenotype of GBM. This corresponds with decreases in GBM cell proliferation and metabolic activity and an induction of GBM cell senescence. Most importantly, we observed that Gli1 PEI-SNAs impair the self-renewal capacity of GBM cells and substantially improve neurosphere response to low doses of TMZ. These results demonstrate that Gli1 PEI-SNAs can reduce GBM resistance to therapy, which is a major hurdle to eradication of this disease.

Overall, this thesis advances the field of RNAi for GBM by showing that Gli1 is an impactful target for GBM therapy, and by developing novel siRNA nanocarriers to target this gene in GBM cells. With continued in vivo investigation and nanocarrier optimization, Gli1-targeted RNAi therapeutics have great potential to alleviate drug resistance and recurrence for GBM patients